Processing of hydrocarbons



United States PatentO Development Corporation, New York, N. Y., a corporation of Delaware No Drawing. Application March 30, 1951,

I Serial No. 218,546

Claims. (Cl. 1953) This invention relates to the production of biosynthetics from hydrocarbons and more particularly to the production of fat-like saponifiable'materials from hydrocarbons by the action of molds, yeasts, and other microorganisms, the invention being an improvement over the invention disclosed in the copending application of R. G. Harris and R. J. Strawinski filed August l7, 1950 bearing Serial No. 180,092 and entitledProcessing of Hydrocarbons.

It has been known for some time that such microorganisms have the ability to attack-ormetabolize some materials such as carbohydrates. Since carbohydrates are relatively expensive raw materials and in most cases, of greater value than the products produced therefrom, other raw materials have been sought. A considerable amount of work has been done with hydrocarbons but to date it has not been generally successful and has not been on a practical basis, apparently because of the failure to appreciate certain factors as hereinafter described. Probably the most discouraging aspect of the hydrocarbon work done heretofore is the fact that the principal products have'been carbon dioxide and water. Apparently, this has been due to a studied adherence to established methods of procedure and a failure to appreciate the possible effects of radical departures therefrom.v

Accordingly, it is an object of this invention to provide novel processes wherein hydrocarbons can be effectively utilized in biosynthesis including the production of relatively high yields of fat-like saponifiable materials and other products. I

Other objects and advantages of the invention will appear from the following description taken in connection with the attached claims.

In brief, it has been discovered that under certain conditions, microorganisms such as molds, yeasts and the like can be caused to grow quite rapidly upon relatively inexpensive substrates such as hydrocarbons in an emulsified phase with the production of relatively large yields of desirable products including fat-like saponifiable materials, and other products. Such conditions include such factors as the selection and maintenance of a proper pH range and temperature range, the selection of proper nutrient salts, and the selection of a proper emulsifying agent. In some cases, the selection of predetermined concentrations of both salts and substrates is desirable.

Broadly stated, the practice of the invention involves the use of a nutrient salt solution such as a solution of the mineral salts type with a pH in a selected range and the use of a hydrocarbon, the use of an emulsifying agent, preferably of a type capable of also functioning as a phosphorylating agent, the inoculation of 'the mixture with a selected microorganism or microorganisms, the maintenance of the mixturewithin predetermined temperature and pH ranges for a time sufficient to effect the desired biosynthesis, and the recovery of the desired products.

Any type of hydrocarbon can be utilized in the process. Good results can be obtained with complex hydrocarbons such as crude oil, kerosine, paraifin wax, naphtha, gasoline, gas oil, lubricating oils and bottom sediments from oil tanks. Of the above, even better results can be secured with certain selected cuts. For instance, a kerosine water white oil, sold by The Texas Company under. the trade name Crystalite and having a B. P. range of 178= C.-261 C. can be fractionated into the following cuts:

Refraction Specific out Index Gravity Of the above cuts of kerosine, Cut D will usually give the best yield and product as expressed in grams of mold mycelia. Cut D will usually give a yield of over twice as much as Cut A, about 50% more than Cut B and about 33% more than Cut C.

Commercial stove oil, having a B. P. range of 167 C.

. to 277 C. can be similarly fractionated by ASTM distillation methods into the following four cuts:

o Refraction Specific out Index Gravity Of the above, Cut I will usually give a yield of about three times the yield of Cut F measured in mold mycelia, of about twice the yield of Cut G and about 1% times the yield of Cut H.

Relatively simple hydrocarbons such as isopentan'e, n-pentane, 2-2-dimethylbutane, 2,2,4-trimethylheptane, n-heptane, isoheptanes, isoheptenes, isooctanes, isooctenes, 2,3-dimethylpentane, cyclopentane, butylbenzene, 2,2,5 trimethylhexane, 2,3 dimethylpentane, Z-pentane, methylcyclopentane, and l-pentene can also be used as well as hexane.

Microorganisms suitable for use include molds, yeasts and the like. Typical examples are molds of the genus Penicillium such as P. rocqueforti, P. glaucum, P. chrysogenum, P. patulum, and notatum, molds of the genus Aspergillus such as A. fumigatus (strain TDC #946), A. carbonarius, A. niger, A. flavus and A. terreus. Typical yeasts include Monilia murmanica, Monilia sitophile and Saccharomyces. I

The majority of the above are available from the American Type Culture Collection. One such mold that igsssgitable for use is unidentified except for the number One of the best microorganisms for use in the process of this invention is Aspergillus fumigatus (strain TDC No. 946) which has been identified as follows:

Source of isolation.A mixed sample composed of soils taken from areas in which oils were undergoing decomposition and water taken from sumps, tank bottoms, and waters which had been in contact with refinery wastes; decomposing asphalt, Wax and used motor oil were included in the sample.

Date of isolation.-October 1, 1947.

Location of isolatiomfiSignal Hill Laboratory, The Texas Company, Long Beach, California.

Media used for isolati0n.A hydrocarbon-mineral salts medium composed of 5% Arabian crude oil (topped) and the following medium at pH 4.5.

Compound:

NaNOg grams per liter 2.0 KH2PO4 0 1.0 MgSO4.7H2O do 0.5 KCl do 0.5 FeSO4.7H2O do 0.01 Sea water milliliters 10.0 Distilled water do 990.0

. scribed the literature indicatethat the mold belongs to the jztm'igatus group of the Aspergilli, the type species of which is Aspergillus fumigatus. A complete description thereof can be found in A Manual of the Aspergilli by Charles Thom, The William and Wilkins Company, Chapter X, pp. .148151.

The character of the nutrient media or mineral salts solution will vary to some extent in accordance with the type of microorganism used and the type if hydrocarbon. Generally, it should contain N, P, K, S, Fe, Mg,

In some cases, the medium may be fortified or enriched with accessory growth factors such as water extract of yeast such as Saccharomyces cerevisiae.

It is to be understood that the character of the medium may also be varied dependent upon the type of product that it is desired to produce in the .most abundance.

As regards the mechanical or contact aspect, the process may bepracticed in a number of different ways. In its simplest form, the growth may be accomplished in a static phase on the surface of the'liquidzmixture. Pre'r-- erably it is practiced as a deep culture process by mechanical agitation such as stirring or shakingin the case of liquids. Inthe case of solid hydrocarbons, contact may be established and:maintained by the method :described in the copending application of Rhett G. Harris,

Serial No. 12,896 filed March.3, 1948 and entitled Rrocesses Involving Action of Microorganisms or Their Enzymes on Organic or InorganicSubstances whereinthe hydrocarbon is adsorbed on a finely dividedzand relatively inert adsorbent material such as. a clay. Where the hydrocarbon is in the gaseous phase, a systenrmay besetup gvherein the gas is recycled through .the inoculated-merum.

The pH with molds may vary from 1 -,to .9. .With yeasts, the pH may vary from 4 to9. its :selection :is governed by the particular microorganisms, the (particular hydrocarbon and the endproducts'desired.

The same-is true of temperature,-.the range beingifrom "C. to '45 C. Theactual time of'incubation,.;usually from 7 to 12 days, is determined by the rate of growth of the microorganisms. of the enzymes, produced by the microorganisms to effect production of the desired products.

The emulsifying agent may be one of the more common and well-known agents for emulsifying hydrocarbon and water but is preferably an agent which is preferentially L soluble in'the hydrocarbon but not in'water. Thus, an emulsion is obtained in Which Water is the dispersed phase. This results in better contact of the'microorganism which is essentially hydrophilic and the hydrophobic source of carbon. Stated otherwise, the resulting mixture is preferably one in which water or salt medium globules are dispersed in hydrocarbon.

It is further desirable that the emulsifying agent be characterized by apparent phosphorylating properties, i. e., capable of entering into the mechanism of fat resynthesis in the animal body.

Calcium stearateis .an example of one of the more common emulsifying agents that might be used but which do not possess .phosphorylating. properties. Apreferred agent, capable. of functioning in both .an emulsifying and phosphorylating.capacity, is lecithin which may be represented by .the.formula:

where R1 aridRz are fatty acid anions, such as oleate,

This is governed 1by-,the:ability i.

4 stearate, etc., and R3 is the quaternary ammonium base choline.

Other monoaminomonophospholipids such as cephalin may be used as well as the diaminomonophospholipids of which the sphingomyelins are typical.

The proportion of emulsifier or phospholipid used in the process does not appear to be particularly critical. Amounts varying from 0.1% by volume of the hydrocarbon to as much as 10 grams per ml. hydrocarbon have been found to produce satisfactory results. The amount of emulsifier used such as lecithin is so small that it would not be used by the microorganism as a carbon source.

As an example of .a method embodying the present invention, lecithin is added to kerosine in a proportion of 0.1 gram of lecithin to 100 ml. of kerosine. The type of hydrocarbon used is not critical nor is the above-indicated proportion of lecithin critical. Proportions as high as l to 10 grams of lecithin to 100 ml. of kerosine have been found to be equally effective.

A suitable sterile mineral salts or nutrient solution is then prepared. Such asolution may vary widely in composition and proportion, the following being typical:

After sterilization, the pH ofsuch a solution is about 5 As already stated, the composition of the mineral salts solution may be Widely variedprovidedthat the following are supplied thereby:

Ammonium ionsuchas ammoniumsulfate or nitrate Magnesium ion Phosphate ion iron and Zinc ion (intraces) Manganese, calcium,vcopper and iodide ions which ap pear to have a stimulatory effect A suitable buffering system to absorb excess .aciditythat may result from the growth process. .Good growths havebeen secured :With an initial pH in the region of 4.5, 5.5 and 6.8.

.A mineral. salts solution, prepared as described above, is addedto the kerosineflecithin. combinationin a proportion of nine,parts mineral salts solution to one part kerosine-lecithin.

The mixture is then stirred andan inoculum including the mycelial :growth and-the .sporesofaseed culture of amold knowntoutilize thespecifichydrocarbon as a source of carbonand energy is added'in an amount of from 10.1% .to 20% by'fvolume. The inoculum is formedsas a thick suspepsion of theorganism in the nutrientrmedium constituting about 10% by volume of the totalcharge. ,The amount of inoculum can be varied widely since .such .variation ,is limiting only upon the periodof .timesubsequently necessary for growth of the myceliato befully established in the desired submerged culture.

While the method has been especially successful with Aspergillus .fumigatus (strain [TDC No. .946), the method is t not restricted .to this particular organism. Any-strain .of any species able-to. utilize the specific hydrocarbon as a source of carbon and energy may be used.

The culture vessel is then held at' a substantially constanttemperature in the range. of about .20 C. to 45 ;'C., preferablyabout 30 3.C.,.and the mixture maintainedinan agitated condition by shaking or stirring. Sterile airor.oxygen,.preferably tat superatmos- Pheric pressure is introducedat .a vigorousrate into the depths of the cu'ltureas through .a porous bubbler.

,After .5.to 10 days operation under these -conditions, the moldmyceliaflis harvested by -mechar ic al ,separation such :as filtration v or .centrifugingfrom the liquid portion of the culture.

As compared with the same method but with the lecithin omitted, the yield of mold mycelia is increased from 200% to 300%.

To recover the fat-like saponifiable material, the mycelia can be dried and a crude extract prepared by chloroform extraction. After removal of the solvent, the residue is treated with approximately two-thirds its weight of potassium hydroxide dissolved in alcohol and refluxed to effect saponification. Following saponification about two-thirds of the solvent alcohol is removed by heating. The residual soap solution is then extracted with petroleum ether to remove the non-saponifiable fraction. Following this step, the aqueous liquor is made strongly acid with a mineral acid and the free organic acids extracted with diethyl ether.

The saponifiable material thus recovered does not consist exclusively of fatty acids of the usual aliphatic character. Some products are found which react chemically very nearly the same as the fatty acids but they are not the usual aliphatic fatty acids such as the straight-chain acids containing an even number of carbon atoms such as C acid (capric), C12 acid (lauric or dodecenoic), etc., such acids being usually obtained from the naturally-occurring fats.

The mixed fatty acids obtained are in the nature of pale yellow solids at room temperature with the characteristic appearance of a fat. A slightly, pleasant, fatlike odor is present and the taste is similar to that of candle tallow or similar stearins. The product melts at about 29.5" C. and has a setting point of 17.5 C. The mean molecular weight is 294, and the iodine number 83.5. Of the common fats or fatty oils, the product most closely resembled the mixed fatty acids from olive oil.

In another method, lecithin is added in an amount of 0.01% the volume of kerosine. About fifteen parts by volume of nutrient medium, prepared as above described, is added to two parts of the kerosine-lecithin mixture and the mixture inoculated with Aspergillus fumigatus. After seven days incubation under agitation and aeration, a yield, dry weight of mycelia is obtained which is almost twice the yield of a control not containing lecithin.

If desired, the mycelia may be processed as described in the afore-mentioned Harris and Strawinski application to recover other products.

From the above it is believed apparent that the present invention provides a novel process for the synthesis of relatively valuable fat-like saponifiable materials and other products in relatively large yields from relatively cheap raw materials, i. e., hydrocarbons. The use of an emulsifying agent, particularly an agent possessing phosphorylating properties, is particularly advantageous in submerged, stirred and aerated culture processes.

Obviously many modifications and variationsof the invention, as hereinbefore set forth, may be made without departing from the spirit and scope thereof and, therefore, only such limitations should be imposed as are indicated in the appended claims.

I claim:

1. The method of synthesizing large yields of fat like saponifiable materials from hydrocarbons which comprises the steps of dispersing hydrocarbon with a phospholipid in an aqueous nutrient medium for microorganisms, inoculating the resultant mixture with a microorganism selected from the group consisting of Penicillium molds, Aspergillus molds, Monilia yeasts and Saccharomyces yeasts, subjecting the inoculated mixture to incubating conditions for a sustained period of time, collecting the resultant solid material and separating synthesized fat-like materials therefrom.

2. The method of claim 1 wherein said phospholipid is lecithin.

3. The method according to claim 1 in which said phospholipid is cephalin.

4. The method according to claim 1 in which said inoculated mixture is incubated at a temperature in the range of 20 to 45 C. and at a pH of about 1 to 9.

5. The method according to claim 1 in which said microorganism is Aspergillus fumigatus.

6. The method according to claim 1 in microorganism is Penicillium notatum.

7. The method according to claim 1 in microorganism is Aspergillus flavus.

8. The method according to claim 1 in which said microorganism is Monilia murmanica.

9. The method of increasing the yield of fat-like saponifiable materials obtained by the action of microorganisms selected from the group consisting of the Penicillium molds, the Aspergillus molds, the Monilia yeasts and the Saccharomyces yeasts on the hydrocarbon in aqueous nutrient medium which comprises the steps of adding a phospholipid emulsifying agent to the mixture of hydrocarbon and nutrient medium.

10. The method according to claim 9 in which said phospholipid is lecithin.

which said which said References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 1,753,641 Beckman Apr. 8, 1930 1,835,998 Giron Dec. 8, 1931 OTHER REFERENCES Bushnell et al., I. Bact., 41, 653-73 (1941). Zobell et al., Bull. Am. Assoc. Petroleum Geol., 27, 1175-93 (1943). 

1. THE METHOD OF SYNTHESIZING LARGE YIELDS OF FATLIKE SAPONIFIABLE MATERIALS FROM HYDROCARBONS WHICH COMPRISES THE STEPS OF DISPERSING HYDROCARBON WITH A PHOSPHOLIPID IN AN AQUEOUS NUTRIENT MEDIUM FOR MICROORGANISMS, INOCULATING THE RESULTANT MIXTURE WITH A MICROORGANISM SELECTED FROM THE GROUP CONSISTING OF PENICILLIUM MOLDS, ASPERGILLUS MOLDS, MONILIA YEASTS AND SACCHAROMYCES YEASTS, SUBJECTING THE INOCULATED MIX TURE TO INCUBATING CONDITIONS FOR A SUSTAINED PERIOD OF TIME, COLLECTING THE RESULTANT SOLID MATERIAL AND SEPARATING SYNTHESIZED FAT-LIKE MATERIALS THEREFROM. 